Use of a bioadhesive composition comprising a polyphenolic protein

ABSTRACT

A non-irritating, non-allergenic and non-toxic bioadhesive composition can be obtained by providing a bioadhesive composition including a polyphenolic protein derived from byssus-forming mussels and, b) a polymer comprising carbohydrate groups. The bioadhesive composition does not contain any enzyme or chemical cross-linking agent. Optionally, the composition may contain an oxidising agent and/or a filler protein. Preferably, the composition is provided as a kit of at least two parts, namely the polyphenolic protein and the polymer comprising carbohydrate groups, respectively. The composition is especially suitable as an adhesive in ophthalmic therapy.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national stage application of InternationalApplication PCT/SE00/02533, filed on Dec. 14, 2000 designating theUnited States of America.

The present invention relates to biodegradable compositions comprisingadhesive, biocompatible polymers and methods used to cover surfaces andto attach structures to eye tissues, such as the cornea. Polyphenolicproteins isolated from mussels, referred to as “mussel adhesive protein”or “MAP”, are used in conjunction with polysaccharides to achieve strongadhesive bonding. The invention also relates to a kit consisting of aMAP preparation, a preparation of polysaccharides which preferably arenegatively charged, and optionally an oxidising agent such as hydrogenperoxide nitroprusside ions or periodate ions, that is used forpreparing a composition for covering and attaching structures to eyetissue, such as cornea.

BACKGROUND OF THE INVENTION

The repair of traumatised structures on and in an eye and its adnexa isoften troublesome. The use of sutures in e.g. the cornea is causingdiscomfort, deformations, distortions and may impair the visual acuity.Other eye components, such as the iris, lens structures and the retinaare difficult or hardly possible to suture or to join with clips andrelated aids. Furthermore, sutures and clips do inevitably induceforeign body reactions and scar formation. The positioning of an implantin ocular structures, e.g. a partial-thickness or a penetratingkeratoplasty or a prosthesis in the cornea, requires the use ofelaborated techniques including the use of haptics to keep it retainedin proper position, which may lead to irritations and adverse reactions.Accordingly, current methods for repairing structures on, at, and in aneye are associated with discomfort and the possibility of inducingpermanent damage to the visual acuity. The use of a composition enablingstructures with wet surfaces to be attached in desired position,remaining adherent for a predicted time period without causing anyopacities in optically important components, or any deformation, scarsor unacceptable foreign body reactions would therefore be highlydesirable.

Polyphenolic proteins, preferentially isolated from mussels, are knownto act as adhesives. Examples of such proteins can be found in e.g. U.S.Pat. No. 4,585,585. Their wide use as adhesives has been hampered byproblems related to the purification and characterisation of theadhesive proteins in sufficient amounts. Furthermore, the requirementfor biocompatible suitable cross-linkers and other additives havelimited their use. Chemicals, such as bifunctional conjugatingcompounds, and enzymes are commonly associated with toxic reactions andother biomedical side effects. Additionally, it is difficult toextensively purify enzymes with retained high activity, avoidingdenaturation and adverse effects on cells, tissues or organs.

Mussel adhesive protein (MAP) is formed in a gland in the foot ofbyssus-forming mussels, such as the common blue mussel (Mytilus edulis).The molecular weight of MAP from Mytilis edulis is about 130.000 Daltonand it has been disclosed to consist of 75-80 closely related repeatedpeptide sequences. The protein is further characterised by its manyepidermal growth factor like repeats. It has an unusual high proportionof hydroxy-containing amino acids such as hydroxyproline, serine,threonine, tyrosin, and the uncommon amino acid 3,4dihydroxy-L-phenylalanine (Dopa) as well as lysine. It may be isolatedeither from natural sources or produced biotechnologically. U.S. Pat.No. 5,015,677 as well as U.S. Pat. No. 4,585,585 disclose that MAP hasvery strong adhesive properties after oxidation and polymerisation, e.g.by the activity of the enzyme tyrosinase, or after treatment withbifunctional reagents. It is very important in biomedical applicationsof an adhesive and coating composition to use bioacceptable andbiodegradable components, which furthermore should not per se or due tocontamination induce any inflammation or toxic reactions. Fillers,including collagens and polysaccharides, have been added to improve themechanical properties in cases when MAP was used to bond tissues andstructures together, further adding to the risk for immunologicalreactions.

It is also previously known that it is possible to use adhesivecompositions based on MAP for ophthalmic purposes. Robin et al.,Refractive and Corneal Surgery, vol. 5, p. 302-306, and Robin et al.,Arch. Ophthalmol., vol. 106, p. 973-977, both disclose MAP-basedadhesives comprising an enzyme polymiser. U.S. Pat. No. 5,015,677 alsodescribes a MAP-based adhesive containing a cross-linking agent andoptionally a filler substance and a surfactant. Preferred cross-linkingagents according to U.S. Pat. No. 5,015,677 are enzymatic oxidisingagents such as catechol oxidase and tyrosinase, but sometimes alsochemical cross-linking agents such as glutaraldehyde and formaldehyde.Examples of fillers are proteins, such as collagen and albumin, andpolymers comprising carbohydrate moieties, such as chitosan andhyaluronan. U.S. Pat. No. 5,030,230 also relates to a bioadhesivecomprising MAP, mushroom tyrosinase (cross-linker), SDS (sodium dodecylsulfate, a surfactant) and collagen (filler). The bioadhesive is used toadhere a cornea prosthesis to the eye wall.

A major problem associated with known MAP-based bioadhesivecompositions, despite the superior properties of MAP per se, is thatsome constituents, in particular the presently used cross-linkingagents, can harm and/or irritate living tissue and cause toxic andimmunological reactions. Chemical crosslinking agents, such asglutaraldehyde and formaldehyde, are generally toxic to humans andanimals, and it is highly inappropriate to add such agents to asensitive tissue, such as the eye. Enzymes, such as catechol oxidase andtyrosinase, are proteins, and proteins are generally recognised aspotent allergens, especially in case they originate from a species otherthan the patient. Because of their oxidising and hydrolysing abilities,they can also harm sensitive tissue. Despite these serious drawbacksassociated with the presently used cross-linkers, it has been regardedas necessary to include them in order to obtain sufficient curing of thebioadhesive.

Accordingly, there is a need for a MAP-based bioadhesive compositionwhich overcomes these drawbacks and hence, does not harm or irritatesensitive tissues such as the eye.

SUMMARY OF THE INVENTION

Now it has surprisingly been found that a non-irritating, non-allergenicand non-toxic composition can be obtained by providing a bioadhesivecomposition comprising a) a polyphenolic protein derived frombyssus-forming mussels b) a polymer comprising carbohydrate groups. Thebioadhesive composition does not contain any enzyme or chemicalcross-linking agent. Optionally, the composition may contain anoxidising agent and/or a filler protein. Preferably, the composition isprovided as a kit of at least two parts, namely the polyphenolic proteinand the polymer comprising carbohydrate groups, respectively.

Definitions

As disclosed herein, the terms “polyphenolic protein”, “mussel adhesiveprotein” or “MAP” relates to a bioadhesive protein derived frombyssus-forming mussels. Examples of such mussels are mussels of thegenera Mytilus, Geukensia, Aulacomya, Phragmatopoma, Dreissenia andBrachiodontes. Suitable proteins have been disclosed in a plurality ofpublications, e.g. U.S. Pat. Nos. 5,015,677, 5,242,808, 4,585,585,5,202,236, 5,149,657, 5,410,023, WO 97/34016, and U.S. Pat. No.5,574,134, Vreeland et al., J. Physiol., 34: 1-8, and Yu et al.,Macromolecules, 31: 4739-4745. They comprise about 30-300 amino acidresidues and essentially consist of tandemly linked peptide unitsoptionally separated by a junction sequence of 0-10 amino acids. Acharacteristic feature of such proteins is a comparatively high amountof positively charged lysine residues, and in particular the unusualamino acid DOPA (L-3,4-dihydroxyphenylalanine). A polyphenolic proteinsuitable for use in the present invention has an amino acid sequence inwhich at least 5% and preferably 6-25% of the amino acid resdues areDOPA. A few examples of typical peptide units are given below. However,it is important to note that the amino acid sequences of these proteinsare variable and that the scope of the present invention is not limitedto the exemplified subsequences below as the skilled person realisesthat bioadhesive polyphenolic proteins from different sources can beregarded as equivalent:

a) Val-Gly-Gly-DOPA-Gly-DOPA-Gly-Ala-Lys

b) Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys

c) Thr-Gly-DOPA-Gly-Pro-Gly-DOPA-Lys

d) Ala-Gly-DOPA-Gly-Gly-Leu-Lys

e) Gly-Pro-DOPA-Val-Pro-Asp-Gly-Pro-Tyr-Asp-Lys

f) Gly-Lys-Pro-Ser-Pro-DOPA-Asp-Pro-Gly-DOPA-Lys

g) Gly-DOPA-Lys

h) Thr-Gly-DOPA-Ser-Ala-Gly-DOPA-Lys

i) Gln-Thr-Gly-DOPA-Val-Pro-Gly-DOPA-Lys

j) Gln-Thr-Gly-DOPA-Asp-Pro-Gly-Tyr-Lys

k) Gln-Thr-Gly-DOPA-Leu-Pro-Gly-DOPA-Lys

As disclosed herein, the term “polymer comprising carbohydrate groups”,relates to a naturally occurring or synthetic polymer comprising aplurality of carbohydrate groups. The polymers can be constituted ofdiscrete carbohydrate groups joined by hydrocarbon chains, butpreferably the polymers are polysaccharides, and still more preferablypolysaccharides comprising charged groups. Examples of suitable polymersare heparin, chondroitin sulfate, chitosan and hyaluronan.

As disclosed herein, the term “pharmaceutically acceptable finefilaments” relates to thin fibres which can be used in sutures,preferably ophthalmic sutures. For the purposes of the presentinvention, the lengths of the fibres should not exceed 15 mm, and aretypically within the range of 1-10 mm.

As disclosed herein, the term “filler protein” relates to a protein thatis optionally added to the bioadhesive composition in order to obtain abioadhesive composition that is adapted to special applications.Suitable filler proteins according to the present invention arecollagen, casein, albumin, elastin, fibrin and fibronectin.

By the term “enzymatic oxidising agent” is meant an enzyme having theability of oxidising MAP in order to promote full or partialcross-linking of MAP and/or polymers and/or filler proteins. Examples ofsuch enzymes according to the state of the art include catechol oxidaseand tyrosinase. A composition according to the present invention doesnot include an enzymatic oxidising agent. By the term “non-enzymaticoxidising agent” is meant a pharmaceutically acceptible oxidising agentwhich, at the doses employed, is non-toxic and non-irritating. Examplesof such non-enzymatic oxidising agents are hydrogen peroxide and sodiumnitroprusside.

By the term “chemical cross-linking agent” is meant a compoundcomprising at least two functional groups that are able to covalentlycouple to MAP and/or polymers and/or filler proteins. Examples of suchcompounds according to the state of the art include glutaraldehyde,formaldehyde, bis(sulfosuccinimidyl) suberate and 3,3′-dithiobis(sulfosuccinimidyl propionate). A composition according to the presentinvention does not include any chemical cross-linking agent.

A composition according to the present invention comprises two mandatorycomponents, namely a) a bioadhesive polyphenolic protein, and b) apolymer comprising carbohydrate groups. It is preferred that thecomposition is supplied as a kit of parts, wherein the above mentionedmandatory components are comprised in separate preparations. Thesepreparations are mixed immediately before use. In the completecomposition, the following concentrations have been found to be useful:

Concentration Component (mg/ml) polyphenolic protein (component a)0.1-50, preferably 0.3-10 polymer with carbohydrate groups 0.1-50,preferably 0.3-30

Optionally, a non-enzymatic oxidising agent can be included. One suchagent is hydrogen peroxide, which typically can be included in an amountof 1-100 mg/ml, preferably about 10 mg/ml corresponding to 1% (w/v).Other such agents are nitroprusside ions and periodate ions. Periodates,such as sodium periodate, are typically in a concentration of 2 mMcounted on the final composition. Furthermore, pharmaceuticallyacceptable fine filaments can be included in an amount of 0.5-40 mg/ml,preferably 1-20 mg/ml of the final composition.

In case the composition is supplied as a kit of parts, each component isprovided in the same or a higher concentration, but the above mentionedconcentration ranges will be obtained upon mixing the componentpreparations with each other in order to prepare the final composition.

The present invention will now be further described in the followingnon-limiting examples.

EXAMPLE 1

Extensively purified mussel adhesive protein ( MAP ) was used, suppliedby BioPolymer Products AB, Floda, Sweden, in 5% acetic acid at aconcentration of 0.9 mg/mL, and stored in the darkness in the cold (˜8°C.). Heparin from swine intestinal mucosa was purchased from SigmaChemical Co., St. Louise, Mo., USA (H 3393). Additional laboratorychemicals were of highest available purity and purchased from Sigma andMerck.

The pH of the MAP solution was adjusted to a slightly alkaline pH,usually to 7,5-8.5 but in additional experiments up to about 9.5. Theexperiments were performed on anaesthetised rats.

The cornea was deepithelialized. A wound was created surgically in thecentre of the cornea, with the aid of a trephine with diameter of 3 mm.The Bowmans membrane and stroma was excised with a knife and finescissors, down to the vicinity of Descemets membrane. A block of cornealtissue was thereby isolated and removed from its original site. At least5 μL of MAP and 3 μL 10 mg/ml aqueous heparin solution was administratedinto the wound cavity and thereafter were the shortly before removedcorneal tissue pieces repositioned into the cavity to test for adhesionand reattachment mediated by the MAP glue. Adherents was achievedbetween the stroma fragments and the wound cavity in the cornea after 5minutes, as very gently tested with the aid of ophthalmic tweezers andan operating microscope. The MAP-glue combination did not add anyobvious extra intensity with regard to the inflammatory reactions duringthe first days. The corneal wound was covered by epithelium within twodays. No adverse effects could be recognised by visual inspection or bymicroscopy that could be related to the MAP composition.

Histopathological examinations of those corneae, that still had stromalfragments attached, after 5 and 7 days revealed that the MAP-heparincombination seemingly did not aggravate the inflammatory response ineither the cornea or in the limbus and conjunctiva as compared to thatin specimens from animals having had a wound cavity in the cornea forthe same time period. There was in places down-growth of epithelialcells into the corneal wound beneath the reattached stromal fragments,but not to that extend that they detached.

EXAMPLE 2

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% citric or1% lactic acid had its pH adjusted to usually to 7,5-8.5, but in someexperiments up to about 9.5. Heparin and laboratory chemicals were ofhighest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. A wound was created surgically in thecentre through roughly half of the cornea, with the aid of a trephinewith diameter of 3 mm. The Bowmans membrane and stroma was excised witha knife and fine scissors, down to the vicinity of Descemets membrane. Ablock of corneal tissue was thereby isolated and removed from itsoriginal site. At least 5 μL of MAP, 3 μL 10 mg/ml aqueous heparinsolution and 2 μL 6% (w/v) aqueous hydrogen peroxide was administratedinto the wound cavity, either once or twice. Thereafter was the shortlybefore removed corneal tissue pieces repositioned into the cavity totest for adhesion and reattachment mediated by the MAP glue. Bonding wasachieved between the stroma fragments and the wound cavity in the corneaafter 5 minutes, as very gently tested with the aid of ophthalmictweezers and an operating microscope. The MAP-glue combination did notadd any obvious extra intensity with regard to the inflammatoryreactions during the first days. The corneal wound was covered byepithelium within two days. No adverse effects could be recognised byvisual inspection or by microscopy that could be related to the MAPcomposition.

Histopathological examinations of corneae with stromal fragmentsattached, after 5 and 7 days revealed that the MAP-heparin combinationseemingly did not aggravate the inflammatory response in either thecornea or in the limbus and conjunctiva as compared to that in specimensfrom animals having had a wound cavity in the cornea for the same timeperiod. There was in places down-growth of epithelial cells into thecorneal wound beneath the reattached stromal fragments, but not to thatextend that they detached due to loss of contact with the cornealstroma.

EXAMPLE 3

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% citric or1% lactic acid had its pH adjusted to usually to 7,5-8.5, in someexperiments up to about 9.5. Chondroitin sulfate and laboratorychemicals were of highest available purity and purchased from Sigma andMerck. The experiments were performed on anaesthetised rats according tothe rules specified by the ethical permissions.

The cornea was deepithelialized. A wound was created surgically in thecentre through roughly half of the cornea, with the aid of a trephinewith diameter of 3 mm. The Bowmans membrane and stroma was excised witha knife and fine scissors, down to the vicinity of Descemets membrane. Ablock of corneal tissue was thereby isolated and removed from itsoriginal site. At least 5 μL of MAP, 3 μL 24 mg/ml aqueous chondroitinsulphate and 2 μL 6% (w/v) aqueous hydrogen peroxide was administratedinto the wound cavity, either once or twice. Thereafter was the shortlybefore removed corneal tissue pieces repositioned into the cavity totest for adhesion and reattachment mediated by the MAP glue. Bonding wasachieved between the stroma fragments and the wound cavity in the corneaafter 5 minutes, as very gently tested with the aid of ophthalmictweezers and an operating microscope. The MAP-glue combination did notadd any obvious extra intensity with regard to the inflammatoryreactions during the first days. The corneal wound was covered byepithelium within two days, although there seemed to be irregularitiesin the epithelial cell layering. No adverse effects could be recognisedby visual inspection or by microscopy that could be related to the MAPcomposition.

Histopathological examinations of corneae with stromal fragmentsattached, after 5 and, for one rat, 7 days revealed that theMAP-chondroitin sulphate combination seemingly did not aggravate theinflammatory response in either the cornea or in the limbus andconjunctiva as compared to that in specimens from animals having had awound cavity in the cornea for the same time period. There was nodistinct down-growth of epithelial cells into the corneal wound.

EXAMPLE 4

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% citric or1% lactic acid had its pH adjusted to usually to 7,5-8.5, in someexperiments up to about 9.5. Hyaluronan (aqueous solution, 10 mg/ml),chitosan (aqueous solution, 10 mg/ml) and laboratory chemicals were ofhighest available purity. The experiments were performed onanaesthetised rats according to the rules specified by the ethicalpermissions.

The surgical procedure was performed as in exampel 1-3. Fairly goodbonding were achieved between the stromal pieces and the surroundingorignal cornea. The achieved results were in agreement with thosereported in Example 1-3. However, histopthalogical examination revealedthat was some down-groth of peithelial cells along the lateral bordersof the implanted stroma, but to a limited extent.

EXAMPLE 5

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% lactic acid hadits pH adjusted to usually to 7,5-8.5, in additional experimentsoccasionally up to about 9.5. Heparin and laboratory chemicals were ofhighest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. The cornea was carefully punched in thecentre with the aid of a trephine with diameter of 3 mm. The tissuecylinder was slowly retracted to at least half of its thickness from theoriginal cornea with the aid of fine-pointed instruments undermicroscopic observation. A solution, prepared immediately before use, of20 μL MAP, 10 μL 10 mg/ml aqueous solution of heparin and 10 μL 6% (w/v)aqueous hydrogen peroxide was applied along the border between the plugand the remaining cornea. The cornea plug was then repositioned andgently kept in position. Adhesion was achieved between the central plugand peripheral part of the original cornea after 5 minutes, as verygently tested with the aid of ophthalmic tweezers and an operatingmicroscope.

Visual inspection and microscopy was performed during 5 days, andoccasionally 7 days. There was an inflammatory reaction in theconjunctiva and blood vessels tended to grow towards the area ofsurgery. The central cornea remained in position fixed to thesurrounding cornea. The cornea was re-epethilialized in 2 days. Therewas edema and slight to moderate opacities in and adjacent to the woundarea. Histopathology of the cornea revealed a slight to moderateinflammation along the zone of surgery. Epithelial cells cover theanterior surface but as well could be detected as strings along partsthe lateral interface of the corneal plug. Leukocytes and macrophagesadhered to the zone of injury, facing the anterior chamber. The achievedresults thus indicate that MAP in conjunction with heparin and anoxidising agent could be used to safely glue a corneal plug back to itssite of surgery.

EXAMPLE 6

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% citric acid hadits pH adjusted to usually to 7,5-9.5. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. Surgery and application of the bondingmixture was performed as in example 5. Adhesion was achieved between thecentral plug and peripheral part of the original cornea after 5 minutes,as very gently tested with the aid of ophthalmic tweezers and anoperating microscope.

The results obtained was in good agreement with those presented inexample 5 for in this case MAP in citric acid and heparin, both withregard to the clinical outcome and the histopatholoy as investigatedafter 5 days.

EXAMPLE 7

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% citric acid hadits pH adjusted to usually to 7,5-8.5. Chondroitin sulphate (24 mg/mlaqueous solution) and laboratory chemicals were of highest availablepurity and purchased from Sigma and Merck. The experiments wereperformed on anaesthetised rats according to the rules specified by theethical permissions.

The cornea was deepithelialized. Surgery and application of the bondingmixture was performed as in example 5. Adhesion was achieved between thecentral plug and peripheral part of the original cornea after 5 minutes,as very gently tested with the aid of ophthalmic tweezers and anoperating microscope.

Visual inspection and microscopy was performed during 5 days, andoccasionally 7 days. There was an inflammatory reaction in theconjunctiva and blood vessels tended to grow towards the area ofsurgery. The central cornea autograft remained in position fixed to thesurrounding cornea. The cornea was re-epethilialized in 2 days. Therewas edema and slight to moderate opacities in and adjacent to the woundarea. Histopathology of the cornea revealed a slight to moderateinflammation in the zone of surgery. Epithelial cells cover the anteriorsurface. However, along the lateral interface of the plug and the corneaonly short, but seemingly wide papilla of epithelial cells could berecognised. In none of the sections examined reached the epithelium morethan about one fourth or one third of the depth of the corneal stroma.Leukocytes and macrophages adhered to the zone of injury, facing theanterior chamber. The achieved results thus indicate that MAP (in citricacid) in conjunction with chondroitin sulphate and an oxidising agentcould be used to safely secure a corneal tissue plug to its site ofsurgery.

EXAMPLE 8

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% lactic acid hadits pH adjusted to 7,5-9.5. Chondroitin sulphate (24 mg/ml, aqeoussolution) and laboratory chemicals were of highest available purity andpurchased from Sigma and Merck. The experiments were performed onanaesthetised rats according to the rules specified by the ethicalpermissions.

The cornea was deepithelialized. The surgery and the treatment with MAP,chondroitin sulphate and oxidizing agent was performed as in example 7.

The results obtained agreed with those in example 7 The achieved resultsthus indicate that MAP (in lactic acid) in conjunction with chondroitinsulphate and an oxidising agent could be used to safely reattach acorneal tissue plug to the cornea.

EXAMPLE 9

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% lacticacid or in 1% citric acid had its pH adjusted to 7,5-9.5. Chitosan (10mg/ml, aqeous solution), hyaluronan (10 mg/ml, aqeous solution) andlaboratory chemicals were purchased to be of highest available purity.The experiments were performed on anaesthetised rats according to therules specified by the ethical permissions.

The cornea was deepithelialized. The surgery and the treatment with MAP,hyaluronan or chitosan and oxidizing agent was performed as in example7. The results obtained with chitosan roughly agreed with those inexample 7, while hyaluronan worked in the acute situation but no longterm results were achieved. The results indicate that MAP in conjunctionwith additional polysaccharides and an oxidising agent could be used toreattach a corneal tissue plug to the cornea.

EXAMPLE 10

Purified MAP (0.9 mg/mL, BioPolymer Products AB) in 5% acetic acid hadits pH adjusted to become alkaline. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. Surgery and application of the bondingmixture was performed as in example 5. Adhesion was achieved between thecentral plug and peripheral part of the original cornea, as gentlytested after 10 minutes with the aid of ophthalmic tweezers and anoperating microscope.

The results obtained was in good agreement with those presented inexample 5 for in this case MAP in acetic acid and heparin, both withregard to the clinical outcome and the histopatholoy as investigatedafter 5 days.

EXAMPLE 11

Purified MAP (0.81 mg/ml, BioPolymer Products AB) in 1% lactic acid hadits pH adjusted to 7.5-9.5. Heparin and laboratory chemicals were ofhighest available purity and purchased from Sigma and Merck. Ophthalmicsutures (10-0) were purchased from Ethicon, cut in small pieces rangingin length from 1-10 mm and eventually added to the MAP solution. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialised. The cornea was carefully punched in thecentre with aid of a trephine with a diameter of 3 mm. The tissuecylinder was slowly reacted to at least half of its thickness from theoriginal cornea with aid of fine-pointed instruments under microscopicobservation. Two small marks were made in the periphery of the centralcornea plug. A solution, prepared immediately before use, of 20 μl MAPsolution, 10 μl aqueous heparin solution, ophthalmic sutures (10 mg/mlof the final composition)and 10 μl 6% (w/v) aqueous hydrogen peroxidesolution, was applied several times along the border between the plugand the remaining cornea. The cornea plug was then repositioned andgently kept in position. Adhesion was achieved between the central plugand peripheral part of the original cornea after 5-10 minutes, as verygently tested with aid of ophthalmic microsurgery instruments using anoperating microscope.

Visual inspection and microscopy was performed during the subsequent 3hours. There was good adhesion between the central corneal plug and thesurrounding residuing cornea. The small defects in the interface zonewere filled by the glue with its filaments.

The achieved results thus indicate that MAP in conjunction with heparin,an oxidising agent and very fine filaments could be used to safely gluea corneal plug back to its site of surgery and to fill and bridgedefects in the tissue.

EXAMPLE 12

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% citric or1% lactic acid had its pH adjusted to usually to 7,5-8.5, but in someexperiments up to about 9.5. Heparin and laboratory chemicals were ofhighest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. A wound was created surgically in thecentre through roughly half of the cornea, with the aid of a trephinewith diameter of 3 mm. The Bowmans membrane and stroma was excised witha knife and fine scissors, down to the vicinity of Descemets membrane. Ablock of corneal tissue was thereby isolated and removed from itsoriginal site. At least 5 μL of MAP, 3 μL 10 mg/ml aqueous heparinsolution and 2 μL 2 mM sodium periodate was administrated into the woundcavity, either once or twice. Thereafter was the shortly before removedcorneal tissue pieces repositioned into the cavity to test for adhesionand reattachment mediated by the MAP glue. Bonding was achieved betweenthe stroma fragments and the wound cavity in the cornea after 5 minutes,as very gently tested with the aid of ophthalmic tweezers and anoperating microscope. The MAP-glue combination did not add any obviousextra intensity with regard to the inflammatory reactions during thefirst days. The corneal wound was covered by epithelium within two days.No adverse effects could be recognised by visual inspection or bymicroscopy that could be related to the MAP composition.

EXAMPLE 13

Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1% citric or1% lactic acid had its pH adjusted to usually to 7,5-8.5, but in someexperiments up to about 9.5. Heparin and laboratory chemicals were ofhighest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

The cornea was deepithelialized. A wound was created surgically in thecentre through roughly half of the cornea, with the aid of a trephinewith diameter of 3 mm. The Bowmans membrane and stroma was excised witha knife and fine scissors, down to the vicinity of Descemets membrane. Ablock of corneal tissue was thereby isolated and removed from itsoriginal site. At least 5 μL of MAP, 3 μL 10 mg/ml aqueous heparinsolution and 2 μL 2 mM sodium nitroprusside was administrated into thewound cavity, either once or twice. Thereafter was the shortly beforeremoved corneal tissue pieces repositioned into the cavity to test foradhesion and reattachment mediated by the MAP glue. Bonding was achievedbetween the stroma fragments and the wound cavity in the cornea after 5minutes, as very gently tested with the aid of ophthalmic tweezers andan operating microscope. The MAP-glue combination did not add anyobvious extra intensity with regard to the inflammatory reactions duringthe first days. The corneal wound was covered by epithelium within twodays. No adverse effects could be recognised by visual inspection or bymicroscopy that could be related to the MAP composition.

1. A method of preparing an ophthalmic adhesive, comprising combining abioadhesive polyphenolic protein and a polymer comprising carbohydrategroups to form an ophthalmic adhesive, wherein said bioadhesivepolyphenolic protein is derived from a byssus-forming mussel comprises30-300 amino acids and consists essentially of tandemly linked peptiderepeats comprising 3-15 amino acid residues, wherein at least 5% of theamino acid residues of said bioadhesive polyphenolic protein areL-3,4-dihydroxyphenylalanine; and wherein enzymatic oxidizing agents orchemical cross-linking agents are not added.
 2. The method according toclaim 1, wherein said polymer comprising carbohydrate groups areselected from the group consisting of heparin, chondroitin sulfate,chitosan and hyaluronan.
 3. The method according to claim 1, furthercomprising adding pharmaceutically acceptable fine filaments, anon-enzymatic oxidizing agent, and a filler protein to said bioadhesivecomposition.
 4. A method for healing performations, lacerations, orincisions, reattaching a retina to the back of an eye, repairing orattaching lenses, and repairing, constructing, reconstructing and/orattaching corneal component parts, comprising: applying an effectiveamount of a composition to said performations, lacerations, orincisions, to reattach the retina to the back of the eye, to repair orattach lenses, and to repair, construct, reconstruct and/or attachcorneal components parts, said composition comprising a component a) abioadhesive polyphenolic protein derived from a byssus-forming mussel,said protein comprises 3-300 amino acids and consists essentially oftandemly linked peptide repeats comprising 3-15 amino acid residues,wherein at least 5% of the amino acid residues of said bioadhesivepolyphenolic protein are L-3,4-dihydroxyphenylalanine, and component b)a polymer comprising carbohydrate groups, wherein said components are inthe form of a combined bioadhesive preparation for simultaneous medicaluse, and wherein said combined bioadhesive preparation does not compriseany enzymatic oxidizing agent or chemical cross-linking agent.
 5. Themethod according to claim 4, wherein said composition comprises apolymer comprising carbohydrate groups selected from the groupconsisting of heparin, chondroitin sulfate, chitosan and hyaluronan. 6.The method according to claim 4, wherein said composition furthercomprises pharmaceutically fine filaments, non-enzymatic oxidizingagents, and filler proteins to said bioadhesive composition.